Selecting for lysis in a temperate phage population

Abstract Temperate phages represent an abundant source of phage diversity, but their capacity for lysogeny limits their therapeutic potential. Here, we asked whether repeated selection for lytic replication could drive a temperate phage toward an obligately lytic lifestyle. We serially propagated a cocktail of three temperate coliphages using a modified Appelmans’ protocol in which phage populations were independently passaged on eight E. coli hosts. Whole-genome sequencing revealed substantial changes in population composition and mutation frequencies. The Uetakevirus phage 1362 was undetectable after the first round, whereas the closely related P2-like phages in the cocktail dominated the evolving populations. Mutations repeatedly accumulated in lysis- and lysogeny-associated genes. Longitudinal genomic analysis revealed rapid fixation of mutations in the integrase coding region, while variants in lysA , lysB , and holin increased in frequency over successive rounds of selection. Protein structure modeling localized selected mutations to regions with potential functional significance. These include the DNA-binding domain of integrase and a surface-exposed region of LysA. Together, these results show that sustained selection for lytic replication can drive rapid and repeatable genomic adaptation in temperate phage populations. Importance Temperate phages could substantially expand the pool of candidates for phage therapy if their ability to enter the lysogenic life cycle can be overcome. While genetic engineering has successfully produced obligately lytic derivatives of temperate phages, experimental evolution has rarely been applied to directly select temperate phage populations towards increased lytic replication. Here we show that repeated selection for lytic replication drives repeatable genomic changes in temperate coliphage populations, including rapid fixation of mutations in the integrase and accumulation of variants in lysis-associated genes. Our study expands the traditional application of the Appelmans’ protocol beyond phage host-range expansion and establishes a framework for directing naturally abundant temperate phages toward increased lytic activity.